The HIV-1 Gag proteins are synthesized as a polyprotein precursor, Pr55Gag, that is cleaved by the viral protease during virus release from the infected cell. Completion of the Gag processing cascade is essential for virus maturation and infectivity. In collaboration with Panacos Pharmaceuticals, we found that 3-0-(3'-3'-dimethylsuccinyl) betulinic acid (PA-457 or bevirimat) potently inhibits HIV-1 maturation by specifically blocking a late step in the Gag processing pathway, the conversion of capsid-SP1 (CA-SP1) intermediate to mature CA. We identified the target of this compound by selecting for and characterizing a panel of drug-resistant mutants. Furthermore, we have recently initiated studies on a structurally distinct compound discovered by Pfizer that also blocks CA-SP1 processing and have identified a partially overlapping panel of resistant mutants. Some of the resistant mutants are highly assembly defective in the absence of the inhibitor and display a striking degree of drug dependence. Efforts are underway to understand the mechanistic basis for maturation inhibitor binding and activity. We hypothesize that defining the structure of the maturation inhibitor binding site(s) will greatly facilitate the development of novel and more potent inhibitors; to this end, we are collaborating with structural biology labs. Finally, we are collaborating with Drs. A. Debnath and M. Summers to define compounds that block maturation by targeting CA. Finally, we are involved in studies aimed at developing derivatives of bevirimat that display greater potency and broader activity against polymorphic isolates of HIV-1. [Corresponds to Freed Project 3 in the October 2011 site visit report of the HIV Drug Resistance Program]